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Abstract This study aimed to evaluate the removal effect of the ProTaper Next system (PTN) combined with manual nickel-titanium Hedström (MNiTiH) files or chloroform on filling materials of mandibular incisors by microcomputed tomography (micro-CT). Sixty-four extracted human mandibular incisors were finally selected and assigned to two groups (n = 32) based on root canal morphology after instrumentation. Two subgroups (n = 16) were formed based on two retreatment methods. The volume of residual filling materials (RFMs) and the surface area covered by RFMs were analyzed by micro-CT, the apical extrusion and the time taken for removing the filling materials were recorded. A smaller percentage of the volume of RFMs and less surface area covered by RFMs occurred in PTN combined with MNiTiH groups and round-shaped canal incisors (P < 0.05). The time taken for removing the filling materials was not significantly different in all groups (P > 0.05). The apical extrusion was significantly less in PTN combined with MNiTiH groups than in PTN combined with chloroform groups (P < 0.05). Neither of the two methods rendered root canals completely free from filling materials. PTN combined with MNiTiH could reduce the apical extrusion and remove filling materials more efficiently than PTN combined with chloroform.